Tobacco use remains a major public health and economic concern, particularly in women who are more vulnerable to the long-term health consequences of smoking than men. Despite the magnitude of the problem, there is a fundamental knowledge gap in our understanding of the mechanisms that promote tobacco use in females. If this knowledge gap is not filled, then reducing tobacco use and developing specialized medications for female smokers will remain largely incomprehensible. The long-term goal of our research program is to identify the mechanisms that mediate tobacco use among different clinical populations that are uniquely susceptible to this problem. The objective of this renewal is to determine the neural mechanisms that promote tobacco use in females. The central hypothesis is that females are more susceptible to tobacco use than males because of stronger rewarding effects of nicotine and heightened anxiety produced by withdrawal from this drug. Our mechanistic hypothesis is that estradiol (E2) promotes the rewarding effects of nicotine and magnifies the stress produced by nicotine withdrawal in females. More specifically, we postulate that sex differences in response to nicotine are modulated within the neural circuits of the nucleus accumbens (NAcc), where dopamine is increased following nicotine administration and decreased during withdrawal from this drug. Thus, females experience greater rewarding effects of nicotine in the presence of E2, which promotes dopamine release in the NAcc. Following repeated nicotine exposure, opponent processes develop to counteract the chronic over-activation of dopamine release. We suggest that the emergence of these opponent processes is evident during withdrawal from chronic nicotine as an increase in the stress hormone, corticotropin releasing factor (CRF) in the NAcc. This increase in CRF levels enhances the inhibitory tone in the NAcc, which results in a decrease in dopamine release during nicotine withdrawal. Thus, females experience greater anxiety during nicotine withdrawal in the presence of E2, which promotes the recruitment of opponent stress systems that suppress dopamine release in the NAcc. The proposed studies reflect a multi- disciplinary approach involving neurochemical, behavioral, and gene transfer techniques to compare sex differences in the rewarding effects of nicotine (Aim 1) and the aversive states produced by withdrawal (Aim 2) because both of these factors are believed to promote tobacco use in females. The approach to studying sex differences involves comparisons of male, female, and OVX female rats. If the removal of ovarian hormones reverses the proposed measures in females, then the role of E2 will be assessed in OVX rats that will receive E2 supplementation and will be tested following E2 or vehicle administration. At the completion of this project, our findings will help us develop a unifying hypothesis regarding the factors that promote tobacco use in females. This exemplifies the significance of this research because a better understanding of the mechanisms that fuel tobacco use in females will lead to more effective treatments to reduce health disparities in women.